Cell-cell adhesion is vital to eukaryotic cell biology, and many of its most diverse manifestations occur between gametes during fertilization reactions. The long-term objective of this research is to characterize the sexual agglutinins that are displayed on the flagellar membranes of plus and minus gametes of the unicellular green alga Chlamydomonas reinhardtii and that mediate a strong and specific adhesion to initiate the mating reaction. The agglutinins are members of the hydoxyproline-rich glycoprotein (HRGP) family, members of which participate in morphogenesis, wound healing, and pathogen defense in higher plants, and their interaction stimulates an activation of intraflagellar adenylyl cyclase and a cAMP-mediated pathway that results in cell fusion. The gene encoding the minus agglutinin has been cloned and almost completely sequenced. It encodes a long PPSPX repeat, found as well in 2 other HRGPs in C. reinhardtii, that forms a membrane-binding domain and a long fibrous shaft, and a large globular domain that forms a head. The proposal has 3 specific aims. 1) Identify and sequence the plus agglutinin, known to be morphologically similar to the minus species, where the similarities and differences between the plus and minus proteins may suggest the location of sex-specific domains. Construct green fluorescent protein (GFP)-tagged agglutinin genes and generate domain-specific antibodies to allow agglutinins to be identified/visualized by real-time confocal microscopy and in quick-freeze deep-etch replicas. 2) Taking advantage of the fact that the agglutinin is the first flagellar membrane protein to be cloned, perform a series of experiments using GFP-tagged wild-type and flagellar-motor-defective mutants to ask whether flagellar membrane protein is transported into the flagellum via the same or different systems than those used to transport axonemal components. Localize the flagellar membrane pool and study its recruitment to the flagellum during flagellar outgrowth. Localize the pool of agglutinin that is recruited to the flagellar membrane with cAMP stimulation and ask whether it differs from the flagellar-outgrowth pool. 3) Determine whether the agglutinin is an intrinsic or extrinsic membrane protein and whether, if intrinsic, it possesses domains important for transport and/or for initiating sexual signaling. Study the disadhesion that accompanies sexual agglutination and occurs at the time of cell fusion, asking whether they are the same or different events and how they occur, the goal being to gain understanding of the adhesion process itself.
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